PROJECT SUMMARY Annually, arboviruses cause morbidity and mortality unparalleled to most other infectious diseases. In fact, billions of people are at risk or arboviral infections creating a large economic burden and imposition on the health systems of many developing countries. With no available vaccine and limited drugs for most arboviral diseases, prevention is essential to disease reduction. Currently, prevention is achieved by sustained vector control; however, with the breakdown or imminent breakdown of many current vector control strategies, we need to develop novel tools to prevent arboviral infection. In this proposal, we aim to improve the technologies available for population replacement strategies, which could be a key tool for the sustainable management of vector-borne disease. While the goal of creating disease, refractory mosquitoes is not novel, we propose to take a novel approach to developing tools for these population replacement strategies. First, we use CRISPR inspired strategies to develop viral sensors that sense and degrade viral RNA. Once these systems are optimized, we will explore using these CRISPR inspired components to create synthetic viral sensors that recognize either viral protein or virus essential host factors and subsequently trigger a signal transduction cascade leading to the production of an antiviral effector. These studies will be performed initially in cell culture models and then transitioned into the dengue, Zika, yellow fever and chikungunya vector, Aedes aegypti. In another approach, we will take advantage of advancements in synthetic RNA switch development in prokaryotic organisms to design and test RNA switches as viral sensors. We will also use the natural architecture of viral RNA switches to develop viral RNA sensors to either outcompete natural viral switches for viral synthesis initiators thereby slowing the replication process, or to actually drive the expression of antiviral effectors. The latter effect would create a negative feedback loop that could potentially halt replication. Additionally, we propose to develop other synthetic tools for mosquitoes, which are inspired by CRISPR, RNA biology and virus regulatory elements. All of these technologies have great potential to improve the tools available to create robust, versatile and consistent antiviral synthetic circuitry in mosquitoes. If successful, these tools would be the first steps towards creating an adaptive immune system for this species, but many of the elements could be used to manipulate any cellular pathway or be used to build novel regulatory pathways. Moreover, for most of these technologies proof-of-principle has only been achieved in an in vitro, prokaryotic or cell culture system. Therefore, it would be a remarkable, albeit difficult, accomplishment to bring these technologies into such an important whole organism system. The potential impact on human health could be tremendous.